Isolation of microorganisms from mixed microbial populations is fundamental to the practice of bacteriology and microbiology. In the past, isolation has involved two general approaches, single cell and fractionation methods. Techniques such as cell plating, cell sorting (Shapiro, H. M., ASM News, 56, 584, 588, (1990) and Shapiro, J. A. 1991, ASM News, 57, 247, (1991) and micromanipulation, operate at the single cell level. These methods rely on spatially separating the mixed population into individual organisms and subsequently allowing each to grow into individual colonies (Johnstone, H. Methods of Microbiology, Vol. 1, Academic Press, Inc., New York 455-471 J. R. Norris and D. W. Ribbons (ed.) (1969)). Single cell techniques are capable of high resolution but tend to select for specific cell types while potentially ignoring a preponderance of other organisms present in the sample. Fractionation techniques, such as selective culture, differential centrifugation, filtration, and adsorption methods operate at the population level. These methods fractionate mixtures into sub-populations based on biological, chemical, or physical differences among the individual groups of organisms (Veldkamp, H., Methods of Microbiology, Vol. 3A, p. 305-361 Academic Press, Inc., New York J. R. Norris and D. W. Ribbons (ed) (1970)). Fractionation approaches tend to have limited resolution. Typically, one or more groups of cell types can be selectively recovered from a mixture using fractionation approaches. However, resolving all sub-populations of a mixture into individual pure fractions is difficult to achieve. Furthermore, fractionation methods can distort the relative numbers or types of organisms in the original sample by selecting for or killing off specific sub-populations. Currently, neither single cell nor fractionation methods provide reliable assessment of the quantitative composition of microbial mixtures. Investigating the composition of mixed microbial populations, thus, continues to be a technical challenge (Herbert, R. A., Methods in Microbiology, Vol. 22, p. 1-33, Academic Press, Inc., New York, R. Grigorva and J. R. Norris (ed.) (1990)). Technology enabling the quantitative separation and recovery of viable microorganisms from mixed populations is a continuing unmet need.
Electrophoresis is the migration of charged substances in a conductive solution under the influence of an electric field. Separation of substances is achieved according to their net surface charge density. Anticonvective media can be interdisposed in the electrophoretic chamber to effect separations based on sieving and other physical interactions. By this means, differences in molecular size and shape can be detected. Anticonvective materials suitable for electrophoresis may include silica gel, glass wool and asbestos, cellulose fibers, sucrose density gradients, cellulose acetate, as well as gel matrices such as gelatin, agar, starch, polyacrylamide, agarose and mixed polymers (Tietz D., J. Chromatogr., 418, 305, (1987)).
Generally anticonvective materials are too restrictive to permit cell migration through the electrophoretic matrix. Therefore, a variety of non-sieving separation electrophoresis techniques has been developed over the years to effect separation of cells in an electric field. These generally use free flow electrophoretic techniques in which cells are migrated through a homogeneous electrolyte buffer solution. Free flow methods may include static techniques such as microelectrophoresis, density gradient electrophoresis or continuous flow electrophoresis techniques such as Continuous Free Flow Electrophoresis (CFFE) and capillary electrophoresis (CE).
Microelectrophoresis involves the direct microscopic observation of visible particles as they migrate in an electric field. The particles, suspended in buffer, are placed in a transparent rectangular or cylindrical chamber. An electric field is then applied across the chamber. The time required for a particle to cover a given distance is measured in a micrometer eyepiece and noted. The results are expressed as mobility per unit field strength (electrophoretic mobility).
Various attempts to characterize electrophoretic mobility of biological cells and viruses using microelectrophoresis methods have been recited in the art. Richmond, D. V., et al., (Advances in Microbial Physiology, 9,1, Ed., A. H. Rose and D. W. Tempest Academic Press, London (1973)) reviewed work regarding electrophoretic mobilities of various classes of viruses, bacteria, trypanosomes, fungi and algae using microelectrophoresis techniques. Work by Bayer, M. E. et al., (J. General Microbiology 136, 867, (1990)), using a Penkem S3000 electrokinetic analyzer, describes the electrokinetic characterization of the charge on Gram-positive and Gram-negative bacteria. Additionally Grotenhuis, J. T. C., et al., (Appl. Environ. Microbiol., 58, 1054, (1992)) teach that seven species of methanogenic bacteria demonstrate significant differences in electrophoretic mobility.
Microelectrophoresis techniques are useful in quantitative measurement of surface charge on individual cells. These studies clearly substantiate differences in surface charge and electrophoretic differences between many microorganisms and different animal cell types. These differences potentially could be useful in separation and recovery of cell populations. However, microelectrophoresis methods do not provide means of quantitative characterization, separation, and recovery of cell populations. Microelectrophoretic measurements require tracking and measuring the rate of migration of individual cells, making the method cumbersome and time consuming. Thus only a small proportion of the total numbers of cells can be characterized. Most importantly, the potential for cell separation is diminished since resolution between cells depends only on differences in one vector force e.g., electrophoretic mobility, and short electrophoresis times.
Other electrophoretic techniques have, however, been used to characterize microorganism populations and explore their separation. Generally these involve a variation of "Continuous Free-Flow Electrophoresis" (CFFE) in which migration is carried out without convective stabilization materials. Separation is achieved in thin films of fluid flowing between two parallel plates. An electric field is applied perpendicular to the direction of electrolyte flow. The electrolytes and sample are pumped through the separation chamber and are collected in an array of collection ports at the opposite end of the separation chamber.
A typical CFFE apparatus comprises a linear separation chamber filled with a separation buffer and bounded on each side by electrodes, across which an electrical potential is maintained perpendicular to the fluid flow. Separation buffer is pumped at a constant flow rate through the chamber from an inlet port to the fraction collecting outlet ports. A mixture of cells is injected at the inlet port of the separation chamber along the edge of the cathode electrode and electrophoretic migration occurs laterally across the width of the separation chamber. The continuous flow of the separation buffer carries the migrated cells to the outlet end of the chamber where they exit into an array of collection ports for analysis (Todd, P., In Cell Separation Science and Technology, 216, A.C.S., Washington, D.C. (1991)).
Continuous Free Flow Electrophoresis (CFFE) has been popular in hematology for the separation of various blood cells and blood components. For example Crawford, N., et al., (In Cell Separation Science and Technology, 190, A.C.S., Washington, D.C. (1991)) disclose the separation of neutrophils from human whole blood using CFFE, whereas Hanse, E., et al., (Electrophoresis, 10, 645, (1989)) discuss the separation of antigen positive and antigen negative human blood lymphocytes and Hannig, K., et al., (Electrophoresis, 11, 600, (1990) demonstrate the use of CFFE for the separation of rabbit, guinea pig and rat erythrocytes.
CFFE methods has also been applied to separation of microorganisms. Fedorikina, O. A., et al., (Mikrobiol. Zh., 48, 83, (1986)) compare the use of three electrophoretic methods in the separation of a mixed population of bacterial cells. The three methods encompassed sucrose density gradient, isotachophoresis and pH gradient electrophoresis. pH gradient electrophoresis effectively separated a mixed population of E. coli and S. aureus into pure populations. Greater specificity has been demonstrated by Hansen-Hagge, T., et al., (Eur. J. Biochem.,148, 24, (1985)) where free flow electrophoresis was demonstrated to be effective in the separation of S. typhimurium lipid A defective mutants from wild type cells. Cells were applied directly to the chamber buffer and fractions were collected over time. This method separated cells deficient in the production of lipid A from the wild type population. Uhlenbruck, G. A., et al., (Zbl. Bakt. Hyg., A 270, 28, (1988)) using a similar "carrier free averting electrophoresis" device for the separation of Group B streptococcus types.
As noted above, CFFE approaches have shown promise for separation and recovery of biological cells. However, CFFE resolution is inherently limited and of marginal. Cell resolution of CFFE relies both on differences in electrophoretic mobility due to differences in surface charge as well as on differences in cell/fluid interactions created when cells interact with the flow of electrolyte fluid. Electrolyte flow in CFFE is laminar and consequently nonuniform across the electrophoretic chamber. At the boundary surfaces of the chamber, electroosmotic flow can further distort the uniformity of electrolyte flow. Furthermore convection currents within the chamber due to Joule heating, vibration and gravitational effects can further distort the uniformity of electrolyte flow with in the separation chamber. As a consequence, cell/fluid interactions are inherently non-uniform, making electrophoretic migration variable and consequently reducing the inherent electrophoretic resolution. Positioning of fraction collecting ports is thus difficult to predict. Attempts to offset these distortions by performing CFFE separations in space (zero gravity) have proved only marginally effective (Todd, P., In Cell Separation Science and Technology, 216, A.C.S., Washington, D.C. (1991)). Potential resolution between cell populations is further restricted by the limited number of fraction collection ports. Limitations on the number of ports that can be designed and mechanically built into the system restricts the number of fractions into which cell populations can be subdivided and limits the ability to discriminate between closely related populations.
Another free flow electrophoretic method is capillary electrophoresis (CE). CE has developed into a powerful analytical separation technique and is particularly useful when sample size is limited since very small sample volumes can be introduced into narrow bore capillary tubes (0.01 to 0.10 mm) for separation. In free solution capillary electrophoresis, the capillary tube is filled with an electrolyte solution, and when an electric field is applied across the capillary, solutes migrate from one electrode toward the other electrode based on the sum of the electrophoretic mobility of the solute and the electroosmotic mobility of the bulk flow of the electrolyte. Because of the small diameter of the capillary, heat is dissipated efficiently, allowing separations to be accomplished at high voltages without distortion from Joule heating. This results in very fast separations without significant loss of resolution. (Jones, H. K., et al., Anal. Chem., 62,2484, (1990); McCormick, R. M., J. Liq. Chromatography, 14, 939, (1991)). However, the high ratio of surface area to electrolyte volume in a capillary can promote both adsorption of solutes onto the capillary walls and distortion of electrophoretic mobility due to electroomotic flow. Both factors can diminish analytical resolution. Typically, these are overcome by chemically passivating the wall of the capillary to diminish electroosmotic flow and reduce adsorption.
Using this technique, Hjerten et al. (J. Chromatogr., 403, 47, (1987)) have shown that movement of the bacterium Lactobacillus casei can be accomplished through a fused-silica tube. The bacteria was sucked into a 115 .mu.m fused silica tube, a voltage applied, and migration occurred toward the anode. The fused-silica tube had been coated with a polymer to suppress electroosmosis and adsorption; as a consequence, movement of the bacteria occurred from the cathode to the anode, based on its negative charge. However, no separation or resolution of bacteria was achieved or disclosed and movement of the bacteria was strictly on the basis of its surface charge. Furthermore, a mechanism for separation and recovery of the isolated bacteria was not disclosed.
Several methods of collecting eluted solutes from CE systems have been reported in the art. For example, Huang et al., (J. Chromatogr. 516, 185, (1990)) have devised a method of fraction collection for capillary electrophoresis which allows continuous contact of the capillary with electrolyte buffer while the analyte is eluting onto a roller covered with filter paper. For collection of materials, this system employs an on-column frit structure to maintain electric continuity between the electrode, cathode buffer reservoir and column buffer. The porous frit structure is constructed about 1-2 cm from the exit end of the capillary tube. This frit allows electrical connections to be made to the capillary, as it is submerged into a buffer reservoir. Two significant drawbacks to the method of Huang are, first, that the porous frit is difficult to construct, and second, sample leaking out of the frit into the buffer reservoir can occur. A further disadvantage of this method is that the system relies on continuous streaking of the sample materials, and does not provide a means of discontinuous programmed deposition of sample fractions in discrete wells or tubes.
In U.S. Pat. Nos. 4,631,120 and 4,631,122, Pohl discloses an apparatus and method for collection of "elemental particles" onto a moving "collecting tape" that are eluted from a porous paper matrix or gel after electrophoretic separation. The system does not provide a means of separation and collection of cells since cell migration is obstructed by molecular sieving effects of the gel and paper separation matrices. Furthermore, the collection tape only moves in one direction and provision is made for X, Y, Z motion of the electrophoretic chamber. Thus the system does not provide for discontinuous sorting and deposition of sample fractions.
U.S. Pat. No. 5,126,025 describes a method and apparatus for collecting CE fractions separated onto a porous layer that retains the solute. A sandwich type collection assembly comprised of three layers is described. The top layer is the porous layer where the sample is deposited. The middle layer is the wetted absorbent layer which maintains electrical continuity. The bottom layer is an electrically conductive plate, attached to the cathode electrode. The exit end of the capillary is in contact with the porous layer, thereby depositing the solute onto the porous layer as the solute is eluted from the capillary end. Collection and deposition of the fractions are accomplished by moving the entire sandwich collection assembly in a circular motion during deposition of the sample. In another mode, the sample can be deposited onto a rotating cylinder. A disadvantage of this apparatus is that the capillary remains in a fixed position. Contact of the capillary and porous layer must be continuous, prohibiting different modes of collection, such as deposition of the solute into tubes or multiwell plates. Thus, the system does not provide for discontinuous sorting or deposition of sample fractions in tubes, microwells or onto solid supports.
There exists a need, therefore, for a method to separate bacterial and other biological cell mixtures into discrete fractions without upsetting the relative distribution of the cells in the original sample, while maintaining the viability of the cells. Furthermore, there is a need to provide a cell collection system enabling individual cells and cell fractions to be sorted and collected in microwell receptacles, tubes, and onto planar supports.
The instant method seeks to meet these needs by providing a novel cell separation method and apparatus for cell collection. Typically, capillary electrophoresis is subject to the complicating forces of high temperature and electroosmotic flow which would be expected to limit viability of the cells and interfere with bacterial and other particle separations (Jones, H. K., et al., Anal. Chem., 62, 2484, (1990); McCormick, R. M., J. Liq. Chromatography., 14, 939, (1991)). The instant method has overcome these problems by utilizing the highly unexpected finding that cells can be separated within a small bore capillary tube by exploiting the resultant effects of two opposing vector forces, electroosmotic flow and electrophoretic mobility. Using this approach, clear separation of cell populations can be achieved.